Brassica species are used as a source of vegetable oil, animal feeds, vegetables and condiments. Brassica plants that are used for vegetable production include cabbage, cauliflower, broccoli, kale, kohlrabi, leaf mustard and rutabaga. However, on a world-wide basis, the most economically important use of Brassica species is for the production of seed-derived, vegetable oils. The predominant Brassica species grown for oil production is B. napus, followed by B. juncea and B. rapa. Seeds of B. napus, B. juncea and B. rapa are referred to as rapeseed. Brassica species that are grown primarily for oil production are often called oilseed rape. In North America, canola, a type of oilseed rape that has been selected for low levels of erucic acid and glucosinolates in seeds, is the predominant Brassica plant grown for the production of vegetable oil for human consumption.
Canola includes three oilseed Brassica species (B. napus, B. rapa, B. juncea) and is grown on over 80 million acres worldwide Canola is a member of the Brassica genus which includes a wide variety of plant species that are under commercial cultivation.
Transgenic canola is currently being cultivated worldwide as a means to solve agricultural production problems. With the development of transgenic canola and other transgenic crops, various countries have instituted regulations to identify transgenic material and their derived products. Polymerase chain reaction (PCR) methods have generally been accepted as the method of choice for transgene detection because of its quantitative and qualitative reliability. This method usually requires amplification and detection of a transgene and a corresponding reference gene, and comparing the quantity of the transgene against the quantity of the reference gene. This system requires a set of two primers and a detection probe specific for the transgene and another set of species specific primers and a probe for an endogenous reference gene.
For the purpose of labeling and traceability, transgene detection assays are developed to meet the requirements of various countries. The European Union's Regulation 619/2011 specifies that the results of detection methods be expressed in transgenic mass fraction with respect to a taxon-specific reference system. The target for the “taxon” specific real-time PCR assay needs to not only be taxon specific, but also quantitatively stable in different genetic backgrounds in order to yield stable testing results.
In most crops, the target species for developing a specific PCR assay is unique, such as for Zea mays, Glycine max, and Oryza sativa. For example, in maize (Zea mays) fields, there are no other closely-related Zea species likely to cross-contaminate a Zea mays field and therefore complicate quantification of maize transgenes. However, canola is quite different.
The Triangle of U (FIG. 1) depicts the evolution and relationship between B. napus, B. rapa, B. juncea and three other Brassica species (Nagaharu U (1935) Genome analysis in Brassica with special reference to the experimental formation of B. napus and peculiar mode of fertilization. Japan. J. Bot 7: 389-452). Through evolution, the 3 base species (B. nigra, B. oleracea and B. rapa) have combined to form three allotetraploid species (B. carinata, B. napus and B. juncea). The three species where canola exists (B. juncea, B. napus and B. rapa) share the A-genome (FIG. 1).
If an endogenous system can be proven to specifically detect the A-genome, it could provide an endogenous reference system for a variety of applications including the relative quantitation of transgenic canola in B. juncea, B. napus and B. rapa. For example, currently most commercial transgenic canola is B. napus, however, an A-specific endogenous reference system could be utilized in detection methods on future transgenics in the other two species (B. rapa and B. juncea). In addition to being able to detect a wide range of varieties of B. rapa, B. napus and B. juncea, the assay must not detect B. nigra, B. carinata, B. oleracea, and other related species that might contaminate a canola grain lot or other major crops, where such cross-detection reduces the accuracy of the assay. Even more, there are other closely-related Brassica relatives that could contaminate canola fields, including, but not limited to, Camelina saliva, Thlaspi arvense, Erucastrum gallicum, Raphanus raphanistrum, Raphanus sativus, and Sinapis arvensis. As such for canola, from a labeling and traceability viewpoint, the challenge is to identify a real-time PCR assay that will be specific to the species that constitute the canola crop.
Several endogenous reference systems currently exist for measuring the relative percentage of genetically modified canola using real-time PCR. However, these systems are not reliable endogenous reference systems (Wu et al., (2010) Comparison of Five Endogenouse Reference Genes for Specific PCR Detection and Quantification of Brassica napus, J. Agric. Food Chem. 58: 2812-2817). They are not specific for the taxon or crop of interest, and they have not been shown to be stable across a globally representative sample within the taxon or crop.
This disclosure relates to methods of detection and quantification that are specific to the Brassica A-genome and does not significantly cross-react with other Brassica species, crops or weedy relatives that could contribute to contamination of a canola field. In addition this endogenous target is stable within each of the three A-genome species when tested on samples from multiple varieties from diverse geographical regions.